Flaviviruses are major human pathogens. They include West Nile, yellow fever and dengue viruses. These viruses result in significant morbidity and mortality each year in temperate and tropical regions ofthe Earth. West Nile virus has spread throughout the US in the last decade, having caused 4052 recorded infecfions and 146 deaths in 2006 alone. We intend to confinue our structural studies ofthe viral life cycle in order to elucidate the processes by which the different viral components are assembled first into immature particles and then metamorphose into mature infecfious virions. We also plan to confinue our structural studies ofthe path by which these viruses infect their hosts, including the inifial host cell recognifion, fusion with the host cell plasma membrane inifiating endocytosis and, finally, the release of the viral genome into the host cell's cytoplasm. A maturafion process that occurs in the final moments before release of infectious virions from a cell is required by most viruses that infect mammals. The immature virions must protect themselves against premature interacfion with the cell's own membranes before being released to infect other cells. Last, but not least, we intend to extend our studies ofthe interacfion between anfibodies and flaviviruses in order to establish the various mechanisms of neutralizafion as an aid to the development of vaccines that do not cause anfibody-dependent enhancement of infecfion. Over the past few years we have learned to produce purified dengue (various strains) and West Nile virus in milligram quantifies of sufficient quality for structural studies. West Nile virus is especially suitable because of its greater stability, but requires bio-safety level 3 facilifies and precaufions. These viruses will be used to produce cryo-electron microscopy three-dimensional reconstrucfions to study immature and mature flaviviruses complexed with various neutralizing antibodies and with cellular receptor molecules. Some of the anfibodies inhibit virus maturafion or fusion at specific intermediate steps in the viral life cycle. We plan to exploit our recent success in determining the crystal structure of the immature virus' heterodimer ectodomain consisting ofthe precursor membrane protein (prM) and envelope (E) glycoprotein. This structure has made it possible to build pseudo-atomic models of immature dengue virus and of a low pH intermediate prior to the release ofthe pr polypepfide and maturafion into infecfious particles. Mutafional and structural studies will now permit determinafion of the amino acids that control the very large conformational changes that occur when the virus matures into infecfious particles. These studies are essenfial for developing anti-viral and vaccine strategies to establish viable defenses against natural epidemics or bio-terrorist attacks based on using fiaviviruses as a weapon. RELEVANCE (See instructions): Flaviviruses, which include West Nile, yellow fever and dengue viruses, are significant human pathogens that give rise to major concerns for human health the Worid over. Dengue virus alone causes 50 million or more cases of infecfion woridwide each year, resulfing in 24,000 deaths. We propose in-depth studies of the structural changes that occur in the assembly pathway and infecfion process during the life cycle of flaviviruses, in particular West Nile and dengue viruses. Such informafion is essenfial for the development of anfiviral drugs and vaccines and for determining the best response in the event of any possible epidemic.